Over the years, chemical analysis techniques have progressed. In the early days, analysis procedures included liquid chromatography, which relates to a process for isolation had purification of compounds. In the early days, commercial liquid chromatographic methods were plagued with difficulties for a laboratory scientist. Later on, certain chemical separations used techniques such as open-column chromatography, paper chromatography, and thin-layer chromatography. Unfortunately, certain limitations existed with these techniques. For example, these chromatographic techniques were often inadequate for quantification of compounds and resolution between similar compounds. Accordingly, pressure liquid chromatography was developed. Such pressure chromatography improved flow through time, which often reduced purification times of certain compounds being isolated by the column approach. Unfortunately, flow rates were often inconsistent, See, Analytical Chem. Volume 62, Number 19, Oct. 1, 1990.
Accordingly, high pressure liquid chromatography (“HPLC”) was developed to resolve some of these limitations of prior techniques. High pressure liquid chromatography improved development of column design and materials. Improvements to high pressure liquid chromatography improved separation between certain compounds, which were similar. More recently, computers and other automation have been added to HPLC for efficiency. Other techniques rely upon electro-osmotic forces for HPLC. An example of such HPLC has been described in U.S. Pat. No. 6,572,749, titled Electrokinetic High Pressure Hydraulic System (herein “the '749 patent”). The '749 patent generally claims an apparatus for fluid flow using electro-osmotic force applied to an electrolyte. The electro-osmotic force is used for an HPLC application. Unfortunately, numerous limitations exist with the electro-osmotic technique for HPLC. For example, electro-osmotic flow using electric fields to cause pressure for pumping and/or compressing liquids. In order to achieve a high pressure, a high voltage, such as 3000 volts, is usually needed. Additionally, the packing of porous materials inside the microchannel is also desired. Although HPLC has improved over the years, many limitations still exist.
From the above, it is desired to have an improved HPLC technique. A particularly important application is proteomics.
Proteomics, a study of protein structure and function, is a research focus for decades to come as it can allow one to elucidate the fundamentals of life and the molecular basis of health and disease. Analysis of complex protein mixtures usually involves two steps: separation and identification. A method of choice for protein identification is mass spectrometry carried out by electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI). Two separation methods dominate proteomic landscape: 2D gel-electrophoresis (2D-GE) and multidimensional high-performance liquid chromatography (HPLC). An important advantage of HPLC compared to 2D-GE is a simple coupling to MS through ESI.
One of the demands of the fast growing proteomic research is a miniaturization of bioanalytical techniques, see e.g. T. Laurell and G. Marko-Varga, “Miniaturization is mandatory unraveling the human proteome”, Proteomics, (2002), Vol. 2, pp. 345-351, incorporated hereby by reference in its entirety, Lion, N.; Rohner, T. C.; Dayon, L.; Arnaud, I. L.; Damoc, E.; Youhnovski, N.; Wu, Z. Y.; Roussel, C.; Josserand, J.; Jensen, H.; Rossier, J. S.; Przybylski, M.; Girault, H. H. Electrophoresis 2003, 24, 3533-3562, incorporated hereby by reference in its entirety. The miniaturization in liquid chromatography is evidenced by smaller beads, smaller diameter columns, and correspondingly smaller flow rates which have led to higher resolution, increased sensitivity, and faster separation.
An integration of LC-ESI on a single chip still has not been yet achieved. For example, a commercially available microfluidic chip by Agilent integrates a trapping column, separation column and electrospray source within a single structure, see e.g. Gottschlich, N.; Jacobson, S. C.; Culbertson, C. T.; Ramsey, J. M. Anal Chem 2001, 73, 2669-2674; Fortier, M. H.; Bonneil, E.; Goodley, P.; Thibault, P. Anal Chem 2005, 77, 1631-1640, both incorporated hereby by reference. However, the Agilent chip is still connected to a conventional LC system to deliver the gradient.
An integration of a complete LC-ESI system, including a pumping system, on a single chip is highly desirable for several reasons. First, an integration of a complete LC-ESI system on a single chip allows one to virtually eliminate a dead volume and, thus, improve an efficiency of LC analyses. Second, an integration of LC-ESI system on a single chip allows one to seamlessly integrate on-chip sensors which can improve the system's reliability and control. Third, miniaturization and integration of an entire LC-ESI system on a single chip can make the system portable. Fourth, miniaturization and integration of an entire LC-ESI system on a single chip can lead to a decrease in power consumption. Fifth, a full integration of a complete LC-ESI on a single chip can greatly reduce cost of the system. Additional advantages for integration are present.